1Department of Molecular Cell Biology, University of Groningen, 9751 NN Haren, The Netherlands.

Erratum in

Mol Biol Cell. 2008 Aug;19(8):3613.

Abstract

Generation of a phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P(3)] gradient within the plasma membrane is important for cell polarization and chemotaxis in many eukaryotic cells. The gradient is produced by the combined activity of phosphatidylinositol 3-kinase (PI3K) to increase PI(3,4,5)P(3) on the membrane nearest the polarizing signal and PI(3,4,5)P(3) dephosphorylation by phosphatase and tensin homolog deleted on chromosome ten (PTEN) elsewhere. Common to both of these enzymes is the lipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)], which is not only the substrate of PI3K and product of PTEN but also important for membrane binding of PTEN. Consequently, regulation of phospholipase C (PLC) activity, which hydrolyzes PI(4,5)P(2), could have important consequences for PI(3,4,5)P(3) localization. We investigate the role of PLC in PI(3,4,5)P(3)-mediated chemotaxis in Dictyostelium. plc-null cells are resistant to the PI3K inhibitor LY294002 and produce little PI(3,4,5)P(3) after cAMP stimulation, as monitored by the PI(3,4,5)P(3)-specific pleckstrin homology (PH)-domain of CRAC (PH(CRAC)GFP). In contrast, PLC overexpression elevates PI(3,4,5)P(3) and impairs chemotaxis in a similar way to loss of pten. PI3K localization at the leading edge of plc-null cells is unaltered, but dissociation of PTEN from the membrane is strongly reduced in both gradient and uniform stimulation with cAMP. These results indicate that local activation of PLC can control PTEN localization and suggest a novel mechanism to regulate the internal PI(3,4,5)P(3) gradient.

Effect of the PI3K inhibitor LY294002 on chemotaxis of AX3 and plc-null cells. Chemotaxis of wild-type (open circle) and plc-null (open triangle) cells was measured to different concentration of cAMP. To investigate the role of PI3K in chemotaxis, both wild-type (closed circle) and plc-null (closed triangle) cells were incubated with the PI3K inhibitor LY294002 at a concentration of 50 μM. The results show the means and SE of the means of three independent experiments.

PLC regulates cAMP-mediated PI(3,4,5)P3 formation during chemotaxis. To investigate the effect of PLC on PI(3,4,5)P3 levels, the PI(3,4,5)P3 detector PHcracGFP was expressed in wild-type, plc-null, and PLCOE cells. (A) Confocal images are shown for cells stimulated with a micropipette, containing 10−4 cAMP from the right. The figures show a representative cell for each case. Bar, 10 μm. (B) Chemotactic properties of cells stimulated with a micropipette, containing 10−4 cAMP. The chemotaxis index (black bar) and speed (gray bar) were calculated from three independent movies; data shown are the mean ± SD of the mean (the difference between control and PLCOE is significant for chemotaxis, *p < 0.05 and for speed, ***p < 0.001, t test).

PLC regulates cAMP-mediated PI(3,4,5)P3 signaling. (A) Membrane translocation of PHCRACGFP in AX2, HAD236 (plc-null), and PLCOE cells after uniform stimulation with 1 μM cAMP. Bar, 10 μm. Quantitation of PHCRACGFP in the cytosol is shown in the bottom. Values are the mean of three independent experiments. Error bars represent SD (the difference between AX2 and plc-null or between AX2 and PLCOE is significant for ***p < 0.005). (B) PKB phosphorylation in wild-type and plc-null cells. Cells were cAMP pulsed, and then they simulated with 1 μM cAMP. Samples were removed at the times indicated and lysed directly into SDS gel loading buffer. Extracts were then fractionated by SDS-PAGE and analyzed by Western blotting probing with a phospho-threonine–specific antibody. The arrows indicate a protein of ∼60 kDa that in wild type was rapidly induced in response to stimulation, indicating, corresponding to the phosphorylation of PKB/Akt, described by others (Lim et al., 2005). This phosphorylation was blocked by pretreatment with the PI3K inhibitor LY294002, and it was strongly reduced in plc-null cells. The blot shown is representative of three independent experiments.

Distribution of PI3K-GFP and PTEN-GFP in AX3 and plc-null cells. To investigate the mechanism by which PLC regulates cAMP mediated PI(3,4,5)P3 accumulation, the distribution of PI3K-GFP and PTEN-GFP were analyzed during chemotactic stimulation. (A) Confocal images are shown for AX3 and plc-null cells expressing PI3K-GFP (top) and PTEN-GFP (bottom). Cells were stimulated with cAMP by a pipette that is positioned at the right. Bar, 10 μm. (B) Translocation of PTEN-GFP in Ax2 and HAD236 (plc-null) cells after uniform stimulation with 1 μM cAMP. Quantitation of the translocation of PTEN-GFP into the cytosol upon uniform cAMP stimulation is shown (bottom). Values are the mean of three independent experiments, and error bars represent SD (*p < 0.05, **p < 0.01, t test).

plc-null cells are defective in aggregation at low cell density. Wild-type (Ax2) and two independent plc-null clones were allowed to develop on nitrocellulose filters at different cell densities (cells per square centimeter). Images were taken after 20 h, and they are representative of multiple experiments.

Model of PLC-mediated PI(3,4,5)P3 formation at the leading edge of chemotaxing cells. The model contains of two regulatory loops: first, a PLC-regulated PI(4,5)P2/PTEN loop (indicated in red) inhibiting PI(3,4,5)P3 degradation; and second, a PI3K/F-actin loop (indicated in green) providing PI(3,4,5)P3 formation and pseudopod extension. cAMP binding to the cAR1 receptor leads to the activation of Gα2 and subsequently in activation of PLC at the leading edge of the cell. Activation of PLC results in the degradation of PI(4,5)P2 at the leading edge and translocation of PTEN to the rear of the cell. The resulting gradient of PI(4,5)P2/PTEN mediates an opposite PI(3,4,5)P3 gradient. PI3K and PTEN are localized at sites of their effector; hence, PI(3,4,5)P3 induced F-actin and PI(4,5)P2, providing stabilization of the gradient and pseudopod extension from the leading edge.